Extraction of penicillin



Patented July 31, 1951 Bettew, WestChester, Pa assignors to Wye th Incorporated, Philadelphia, Pa, a. corporation of Delaware No Drawing. .Applioation February 26, 1944,

, Serial No. 524,144

1 This invention pertains e ll n f c ude roth co tain nethea ame.

a d r c arly ner i stoame hod 9i has en ing he ep at en of he or anic .eolventla rn n g the enic llin. from th aqneeuebro h.

aye r nethe r ees oi extraction... a

In the production of penicillin, it is customary to extract the penicillin from its crude broth culture medium by means of a water-immiscible organic solventsuchgas amyl acetate or chloroform. This extraction serves not only to concentrate and purify he p n cill n ut a so serves to transfer the penicillin to an environment in which the penicillin is more stable. To extract the penicillin effectively it is necessary,

that therwater-immiscible organic solvent be agitated thoroughly and rather vigorously with the crude broth culture medium. Unfortunatey h agitation gene al y res ts n the a ueous and, orga ic solvent la s orming a p sist entemulsion that, breaks" into two layers exmel slowly; in fact s m mes ven af er prolonged standing an emulsion interphase persists between the two layers. Such a condition is undesirable both from the standpoint of slowing d t e production of penicill also rom the standpoint of loss of penicillin because of incomplete separation of the aqueous layer.

One object of the present invention is to facilitate the extraction of. penicillin-from its crude broth culture medium.

Another object of the inventionis to speedup the production of penicillin without increasing the amount of production equipment required.

A specific object of the invention is to break more quickly into separate layers the emulsion to. th extraction of now 12. cl ims; .(01. 260-2391) break of the emulsion to form two sharply defined layers consisting of an organic solvent layer containing extracted penicillin and an aqueous layer from which the penicillin has been extracted." Preferred agents of sucha type are the sulfated high alcohols sold in commerce under,

the tr d ames 111111011101 .05, nup n Tergitol #7 (sodium sulfate derivative of -3.9=- di hyltridecanol-B); and lfTerlgitol #4..- other p i r d' aeent fin -c1101 WX which:

' is the sodium salt of ahlgherhetroleum aliphatic hydrocarbon sulionate. A great variety of emulsifying agents, however; may. be. used llicii 1. ly to speed the breaking--ofthe emulsion into two sharply defined layers. Thus there mayv lee-- mentioned soaps, such-as the ammonium soap of cocoanut oil'fattyf acidsand'f'soldunder thetrade name of"Wecoline A, or triethanolamine oleate (either in proportions of one to one of triethanol'amine and oleic-acidor in proportions of one to two). In the claimsethefexpression ammonium soaps is intended to include substituted ammonium soaps such as triethanolew seene ma il sulf iate an such, ae th t so d th am o cesarean Su ate emulsi-fying agent one may add a condensation .product of formaldehyde a naphthalene .1:

that forms when a water-immiscible or anic solvent is agitated vigorously with the crude culture broth containing the penicillin.

A more specific object of the invention is to furnish an agent that in small amounts is without harmful effect on the penicillin but that is effective in braking more quickly into separate layers the emulsion that forms when a water-immiscible organic solvent is agitated vigorously with the crude culture broth containing the penicillin.

We have found that the addition of a small amount of an emulsifying agent surprisingly achieves the above objectives and others that will. become apparent hereinafter in the specification. Emulsifying agents of the type capable of producing oil-in-water emulsions and of lowering the surface tension of water even in the presence of acid are especially useful in promoting a quick fonieacid sold unde ena ri o ana .,,1 PW., .Qther su table emuls i e a ents are Emulphor Turkey red 011 and Product a.

which is cetyl. triinethyl ammonium bromide;

emulsifying agent. necessary to produce the desired result is critically important foreach emulsifying agent selected and that the amount is adde .tn femiils fieat pn ii ltiee a e ria-' menteel e henha d reasesir or; cc. to

satisfa t rnresult greater amq T13... 17-: 'i'f iu The following table serves to illustrate' speci-e ficallynthew. effectiveness of typical emulsif in agents. In securing these data, a crude culture broth containing penicillin was cooled to 2 to 4 C., filtered through cheese cloth, and acidified to a pH of 2.2-2.4 with 10% phosphoric acid. For

tion of the emulsifying agents may be enhanced by the addition of a small amount of an electrolyte, for instance, that of ,Tergitol No. 7 by adding a drop of 10% alum solution. Other In such cases,

For conveneach test 10 cc. of the cold, filtered, and acidified electrolytes, preferably of thepolyvalent type, are broth were placed in.a 25cc. stoppered graduated useful for the same purpose. z, i cylinder together with '7 .5 cc. of amyl acetate and We prefer to carry out the extraction of the the requisite amount of emulsifying agent. In crude penicillin culture broth with amyl acethe case of the Control, .5 cc. of water was added tate. Emulsification difficulties are encountered to compensate to a degree for the volume added also, however, in the case of other suitable organic to the other tests in the form of emulsifying solvents, such as chloroform, ethylene dichloride, agents. After shaking all tests the same length butyl acetate, ethers, and water-immiscible keof time to form emulsions, readings were taken tones, such as diisopropyl ketone. at time intervals of 5, 10 and minutes of thethe separation of the emulsion into two sharply volumes of the clarified aqueous portions of the 15 defined layers of'aqueous solution and organic solemulsions. l I vent extract may be. hastened by the presence 01' Table I a small amount of an emulsifying agent. Thereafter the two layers may be separated in any Volume clear aqueous desirable conventional manner or if desired the liquid Separated from emulsion may be passed directly through a Emmsifym A t 17 s 1 ti 1 1 11 151011 after stand- Sharples SuperCentrifuge to separate off the org gen 0 Qns g ganic solvent extract layer without waiting for 5 min mm mm layer formation to occur. If the latter procedure 15 followed, the separation of the organic solvent I Cc Cc Cc 5 extract proceeds much more smoothly in the pres- Control; L 5 5 0 once of the emulsifying agent that in its absence. :Efigitolx v 3.25 5.5 0.5 Almost every type of crude penicillin culture E 2.0 5 5.0 liquids gives rise to emulsification difficulties and; ag 2.2 in all such cases the teachings of the present 30 invention may be applied with advantage. 5.0 0.5 7. typical penicillin crude culture media may be 3, M mentioned the Modified Czapek-Dox consisting 10cc g$ of .5 gm; hydrated magnesium sulfate, 3 gm. of Emm h i :.:5 6 i::: 1 1 6 sodium nitrate, 1 gm. of potassium dihydrogen Wee 0line"A 1 00 2 5 4 5 5' phosphate, .5 gmqpotassium chloride, .01 gm. of I 1 1 hydrated ferrous sulfate, 40 gm. of glucose, and Dummy, cc water sufiicient to make one liter, or a medium Tyiethanolamme 1 'jgfiafifg' igfi'i consisting of 40 cc. of corn steep-liquor, gm. of M 6 25 lactose, 3 gm. of sodium nitrate, .5 gm. of pri- 'i.00 cf: 21:; 5.0 6125 40 mary potassium phosphate, .01- gm. of zinc sulifig fg 1 pt mag-618m acid-f fate, .25 gm. of hydrated magnesium sulfate, and

it-2.0 cc 3.0 5.5 1.0 water sufiicient to make one liter. ience the invention has also been described in The type, manufacturer or distributor, and terms of extracting a crude penicillin broth culchemical characterization of the emulsifying tulei However the additionof emulsifying e ts agents used are indicated in the following table: is generally applicable to minimize 'difficulties Table II Iiai'n'e of Agent Kind of Agent Manufacturer or Distributor Chemical p iti Tergitol #7 Anionic-.." (larbide'dz Carbon Chem. Corp Sodium Salt of suliated i y fl w l-fi' Alkanol WXN -.do E. I. du Pont de Nemours & Co. sodiliflm salt of a higher petroleum aliphatic hydrocarbon Pro'ductQ Cationic; .do rr i in yrcetylammonium bromide. Aerosol O'I Anionic.--" American Cyanamid & Chem. Corp. Dioctyl ester of sodium sulfo-succinic acid. Emulphor 1L-.. Non-ionic"- General Dyestufi Corp Higher fatty poly ethers of the formula:

I R-COO(CHOHO),.CHGH2OH Wecoline A Anionic Wccoline Products, Inc A fractionated eocoanut oil, rich in lauric acid. During v fractionation most ofthe lower boiling compound fatty acids have been removed. Tanasol PW... do Jacques Wolfe & Co Sodium salt of di-sulfo di-naphthyl methane. Duponol ME... .do ,E. I. du Pont de Nemours dz Co Sodiumlauryl sulfate.

Or described somewhat differently, at the end of the five, ten and fifteen minute periods, 54.5%. 68%, and 78% of the aqueous layers had sharply separated from the amyl acetate layer in the case wherein 1.0 cc. of Alkanol WXN had been used as compared to 14.3%, 33.2%, and 47.5%, respectively, in the case of the Control. While the above data are given on the basis of small scale experiments, a corresponding and sometimes greater improvement is obtained when the emulsifying agents are added in the same proportion in extracting plant size batches of the crude penicillinbroth. I

"As will be noted from Table I above, the ac-' wherever liquid containing penicillin has a tendency to form an emulsion during extraction. Forinstance, sometimes the crude penicillin broth culture is treated with Darco-G-60 'or similar ad-.- sorbent carbon black to adsorb the penicillin, the

carbon adsorbate eluted with amyl acetate and water, and the eluate extracted with amyl acetate. In such cases emulsion difficulties are likewise encountered and the breaking of the emul-;

sion' may be accomplished by the addition of a small amount of emulsifying agent.

The demulsification activity shown by emulsifying agents upon the emulsion resulting from mixing a water-immiscible organicsolvent with waters-1s a crude penicillin lirothrultuiris rather remarkable and difficultto explain. Qnepossible explanationisthat the added emulsifyin'gfiagent reacts with the polyvalent electrolyte present in the crude culture penicillin broths to form acom pound that nullifies not, only the emulsifying character of the added emulsifyingagent butf'also the emulsifying character of" the unknown emulsifying agent inherently present infth'e crude penicillin culture broth. i r f As many apparently widlyfldiffereiit embodiments of this invention may bemade without departing from the spirit and scope thereof, it is to be understood that we do not limit ourselves to the above description except as defined in the appended claims.

We claim:

1. In the process of extracting penicillin from a crude acidic aqueous culture liquid with a water-immiscible organic solvent, the improvement which comprises adding thereto, as a demulsifier, a small amount of an emulsifying agent substantially inert to penicillin and having the properties of reducing the surface tension of water and promoting the formation of oil in-water emulsions in aqueous media having an acid reaction, said emulsifying agent consisting essentially of a material from the group consisting of (1) higher alcohol sulfates. (2) sulfonated higher hydrocarbons, (3) higher alkyl esters of sulfosuccinic acid, (4) long-chain-alkyl tri-lower-alkyl quaternary ammonium halides, and (5) ammonium soaps of higher fatty acids, thus accelerating separation of the organic solvent extract from the aqueous phase, and separating the resulting organic solvent extract from the aqueous phase.

2. The process steps defined in claim 1 in which the extraction is carried out at a pH of substantially 2.

3. The process steps defined in claim 1 in which the emulsifying agent is added in an amount within the range 0.003% to 0.1% by weight of the amount of organic solvent employed.

4. In the process of extracting penicillin from a crude acidic aqueous culture liquid with a water-immiscible organic solvent, the improvement which comprises adding thereto, as a demulsifier, a small amount of an emulsifying agent having the properties of reducing the surface tension of water and promoting the formation of oil-in-water emulsions in aqueous media having an acid reaction, said emulsifying agent consisting essentially of an alkali metal salt of a higher alkyl sulfate, thus accelerating separation of the organic solvent extract from the aqueous phase and separating the resulting organic solvent extract from the aqueous phase.

5. In the process of extracting penicillin from a crude acidic aqueous culture liquid with a water-immiscible organic solvent, the improvement which comprises adding thereto a small amount of the sodium salt of the sulfate of 3,9- diethyl tridecanol-B, as a demulsifier, and separating the resulting organic solvent extract from the aqueous phase.

6. In the process of extracting penicillin from a crude acidic aqueous culture liquid with a water-immiscible organic solvent, the improvement which comprises adding thereto, as a demulsifier, a small amount of an emulsifying agent having the properties of reducing the surface tension of water and promoting the formation of oil-in-water emulsions in aqueous media having an acidireactioiil s'aidemulsifying agent mile ggess m a lv f s lfona dhigher petroleum hydrocarbon, thus accelerating separation of the organic solvent extract from the aqueous phase and separating the resulting organic solvent extract from the aqueous phase. 'i 1 7. In the processof extracting penicillinfrom a crude acidic aqueous. culture liquid with a water-immiscible organic solvent, the improvementwhich'comprises adding thereto, as ademul'sifier, a small. amount of an emulsifying agenthaving the properties of reducing the surface tension of water andpromoting the formation of oileinewater emulsions inaqueous media having an acid reaction, said emulsifying agent consisting essentially of di-sulfo di-naphthyl methane, thus accelerating separation of the organic solvent extract from the aqueous phase and separating the resulting organicsolvent extract from the aqueous phase.

8. In the process of extracting penicillin from a crude acidic aqueous culture liquid with a water-immiscible organic solvent, the improvement which comprises adding thereto a small amount of dioctyl ester of sodium sulfo-succinic acid as a demulsifier, and separating the resulting organic solvent extract from the aqueous phase.

9. In the process of extracting penicillin from a crude acidic aqueous culture liquid with a water-immiscible organic solvent, the improvement which comprises adding thereto, as a demulsifier, a small amount of an emulsifying agent substantially inert to penicillin and having the properties of reducing the surface tension of water and promoting the formation of oil-inwater emulsions in aqueous media having an acid reaction, said emulsifying agent consisting essentially of a long-chain-alkyl tri-lower-alkyl ammonium halide, thus accelerating separation of the organic solvent extract from the aqueous phase, and separating the resulting organic solvent extract from the aqueous phase.

10. In the process of extracting penicillin from a crude acidic aqueous culture liquid with a water-immiscible organic solvent,- the improvement which comprises carrying out the extraction in the presence of a small amount of a longchain-alkyl trimethyl ammonium bromide, as a demulsifier, thus accelerating separation of the organic solvent extract from the aqueous phase, and separating the resulting organic solvent extract from the aqueous phase.

11. In the process of extracting penicillin from a crude acidic aqueous culture liquid with a water-immiscible organic solvent, the improvement which comprises carrying out the extraction of the acidic aqueous culture liquid at a pH of about 2 and in the presence of a small amount of a long-chain-alkyl trimethyl ammonium bromide as a demulsifier, thus accelerating separation of the organic solvent extract from the aqueous phase, and separating the resulting organic solvent extract from the aqueous phase.

12. In the process of extracting penicillin from a crude acidic aqueous culture liquid with a water-immiscible organic solvent, the improvement comprising adding thereto about 1 of a long-chain-alkyl trimethyl ammonium bromide,

(References on following page) v. Q REFERENCES CITED n .OTI-IER-REFERENCES "The following references are of record in the Report of Investigations, Dept. of Interior fileot this patent: Bureau of Mines, Serial No. 2,692, June 1925',

Number Name Date Proc d'n s f and Petroleum Con r'e'ss' 1,938,322 DeGroote Dec. 5, 1933 f g g 1938323 De Groote' 5,1933 Condensed Dictionary, page 51, 3rd ed., 1942:

13431815 De Groom 1934 10 Synthetic Organic Chemicals, by Union Car- 1941 ban and Carbide, 11th ed., July 1, 1942; p. 62. 3741 De 1941 'Theory of Emulsions, 4th ed.', 1943, pp. 475- 2,281,'419 De Groote' Apr. 28, 1942 .r

2,338,384 Robinson Jan. 4, 1944 v2,448,790 Foster et a1. Sept. '7, 1948 

10. IN THE PROCESS OF EXTRACTING PENICILLIN FROM A CRUDE ACIDIC AQUEOUS CULTURE LIQUID WITH A WATER-IMMISCIBLE ORGANIC SOLVENT, THE IMPROVEMENT WHICH COMPRISES CARRYING OUT THE EXTRACTION IN THE PRESENCE OF A SMALL AMOUNT OF A LONGCHAIN-ALKYL TRIMETHYL AMMONIUM BROMIDE, AS A DEMULSIFIER, THUS ACCELERATING SEPARATION OF THE ORGANIC SOLVENT EXTRACT FROM THE AQUEOUS PHASE, AND SEPARATING THE RESULTNG ORGANIC SOLVENT EXTRACT FROM THE AQUEOUS PHASE. 